1. Field of Invention
The present invention relates to a reagent for assaying haptens and its application.
Haptens are nonimmunogenic molecules, namely when they are alone they are incapable alone of triggering an immune reaction by antibody production, but are capable of being recognized by antibodies obtained by immunizing animals under known conditions, particularly by immunization with a hapten-protein conjugate.
2. Description of Related Art
Various techniques have been proposed for assaying haptens in a sample, including the so-called competition techniques. These techniques, described for the first time by Yalow and Berson (Nature, 184, p. 1648, 1959) for radioimmunoassay of plasma insulin, were applied to assaying haptens in the early 70s (Gharig H. et al. J. Clin. Endocrinol. Metab., 31, pp. 709-714, 1970; Abraham G. E., Acta Endocrinologica, suppl. 183, pp. 1-42, 1974).
According to these techniques, the hapten to be assayed is placed in competition, for binding to a given quantity of specific antibodies of said hapten, with a known, added quantity of the same, labeled, hapten. After a separation stage in which the free hapten (i.e. not linked to the antibodies) and the antibody-hapten complexes are separated, the quantity of labeled hapten, linked or free, is determined by the activity of the label. This measurement allows one to deduce the quantity of unlabeled hapten present at the outset. In this type of competition assay, the quantity of labeled hapten-antibody complex formed decreases as the quantity of unlabeled hapten is increased in the reaction medium.
According to a first embodiment of this type of competition assay, the hapten to be assayed and the labeled hapten react simultaneously with a predetermined quantity of specific antibodies of the hapten. After equilibrium, the free haptens are separated from the haptens bound to the antibodies. According to another embodiment, the reagents are added sequentially: in the first phase, the hapten to be assayed is added to a predetermined quantity of antibodies such that all the hapten to be assayed is bound. In the second phase, the labeled hapten is added in excess to saturate the antibody sites that have remained free and, after a sufficient incubation time, the unbound labeled hapten is separated.
Various methods for labeling haptens have been proposed. Radioactive labeling, although it offers the advantage of being sufficiently sensitive, has various drawbacks such as the instability of the label and the need for cumbersome equipment, and personnel protection and waste disposal measures, which are substantial constraints, particularly as far as automating the hapten assay test is concerned. Another option is to use an enzyme such as horseradish peroxidase, alkaline phosphatase, or beta-galactosidase as the label. Addition of the specific substrate of the enzyme reveals the quantity of free hapten or hapten bound to the antibody (Van Weemen B. K. and Schuurs A. H. W. M., FEBS Letters, 24(1), pp. 77-81, 1972 or Wisdom G. B., Clin. Chem., 22(8), pp. 1243-1255, 1976). This type of labeling has several advantages such as cost, ease of use, speed of enzyme reaction, absence of specific protective measures, and ability to automate the test.
It is known to specialists however that competition methods using an enzyme-labeled hapten are sometimes insensitive and may in some cases give results with low significance.